2-β-D-ribofuranosylselenazole-4-carboxamide compounds and methods for their production

ABSTRACT

A class of novel 2-β-D-ribofuranosylselenazole-4-carboxamide nucleoside and nucleotide compounds and methods for their production are provided. Compounds of the class typically have pharmacological properties, especially antitumor and antiviral properties, and are well tolerated, being useful, for example, in treating tumors and viral infections in warm blooded animals.

This application is a division of application Ser. No. 463,221, filedFeb. 15, 1983, now U.S. Pat. No. 4,531,001 which in turn is acontinuation-in-part of application Ser. No. 360,968, filed Mar. 23,1982, now abandoned.

DESCRIPTION

1. Technical Field

This invention is directed to noval2-β-D-ribofuranosylselenazole-4-carboxamide nucleoside and nucleotidecompounds having pharmacological activity, especially antitumor activityand antiviral activity, and to methods for their production.

2. Background of the Invention

Control of malignant tumors in man and animals still remains as anunrealized goal. Within the last several decades, understanding ofmalignancy has made significant progress; however, conquering of themalignant disease state has not been realized.

Conventional therapy of both humans and other valuable animal speciesinflicted with malignant tumors presently includes surgical excising ofthe tumor, local radiation therapy of the afflicted animal, andchemotherapy by administration of a chemotherapeutic agent to theanimal. The death of a significant number of patients inflicted withmalignant tumors is attributable not to the primary tumor but instead tometastasis of the primary tumor to secondary sites in the host. If aprimary tumor is detected early, it normally can be eliminated bysurgery, radiation or chemotherapy or combinations of these. Themetastatic colonies of these primary tumors, however, are exceedinglyharder to detect and eliminate and the unsuccessful management of themremains a serious medical problem.

Tumors are normally classified either as benign or malignant. Themalignant tumor is characterized by its ability to invade bothsurrounding tissue and to colonize distant sites via metastasis. Certainorgans are more prone to metastasis than others. Included in this groupwould be the lung, the brain, the liver, the ovaries and the adrenalglands. It has further been expected that both surgery and radiation ofa primary tumor in certain instances actually promote metastasis.

In view of the inability of current cancer therapy to successfullycontrol the malignant tumor and its metastasis, a need for additionalchemotherapeutic agents exists.

Similarly, for the control and management of antiviral infections,agents are currently available, but few are clinically applicable andthese are only narrowly active. In this field, therefore, a need foradditional chemotherapeutic agents also exists, especially for agentsthat have both antiviral and antitumor activity.

SUMMARY AND DETAILED DESCRIPTION

The present invention relates to a class of novel chemical compounds andmethods for their production, which compounds are2-β-D-ribofuranosylselenazole-4-carboxamide compounds of the structure Iand precursors of the structure II ##STR1## wherein R₁ and R₂ are eachhydrogen or acyl, preferably benzoyl or C₁ -C₁₈ acyl, and R₃ ishydrogen, acyl (preferably benzoyl or C₁ -C₁₈ acyl) or ##STR2## Z is--C(NH₂)Se or selenazol-4-(lower alkyl or aralkyl)-carboxylate-2-yl;and, when R₃ is phosphono, physiologically acceptable salts thereof.

Preferred compounds, for purposes of the invention are the following:

2-β-D-Ribofuranosylselenazole-4-carboxamide,

2-(2,3,5-Tri-O-acetyl-β-D-ribofuranosyl)-selenazole-4-carboxamide,

2-β-D-Ribofuranosylselenazole-4-carboxamide 5'-phosphate,

2-β-D-Ribofuranosylselenazole-4-carboxamide 5'-phosphate, sodium salt,

2-(2-O-Acetyl-β-D-ribofuranosyl)selenazole-4-carboxamide,

2-(3-O-Acetyl-β-D-ribofuranosyl)selenazole-4-carboxamide,

2-(5-O-acetyl-β-D-ribofuranosyl)selenazole-4-carboxamide,

2-(2-O-Acetyl-5-0-phosphono-β-D-ribofuranosyl)selenazole-4-carboxamide

2-(3-O-Acetyl-5-O-phosphono-β-D-ribofuranosyl)selenazole-4-carboxamide

2-(2,3-Di-O-acetyl-β-D-ribofuranosyl)selenazole-4-carboxamide,

2-(2,3-Di-O-acetyl-5-O-phosphono-β-D-ribofuranosyl)selenazole-4-carboxamide,

2-(2-O-Butyryl-β-D-ribofuranosyl)selenazole-4-carboxamide,

2-(2-O-Benzoyl-β-D-ribofuranosyl)selenazole-4-carboxamide,

2-(2-O-Butyryl-5-O-phosphono-β-D-ribofuranosyl)selenazole-4-carboxamide,

2-(2-O-Benzoyl-5-O-phosphono-β-D-ribofuranosyl)selenazole-4-carboxamide,

2-(3-O-Butyryl-5-O-phosphono-β-D-ribofuranosyl)selenazole-4-carboxamide,

2-(3-O-Benzoyl-5-O-phosphono-β-D-ribofuranosyl)selenazole-4-carboxamide,

2-(2,3,-Di-O-butyryl-5-O-phosphono-β-D-ribofuranosyl)selenazole-4-carboxamide,

2-(2,3,-Di-O-benzoyl-5-O-phosphono-β-D-ribofuranosyl)selenazole-4-carboxamide,

2-(5-O-Butyryl-β-D-ribofuranosyl)selenazole-4-carboxamide,

2-(5-O-Benzoyl-β-ribofuranosyl)selenazole-4-carboxamide,

2-(3-O-Butyryl-β-D-ribofuranosyl)selenazole-4-carboxamide,

2-(3-O-Benzoyl-β-D-ribofuranosyl)selenazole-4-carboxamide,

2,5-Anhydro-3,4,6-tri-O-benzoyl-D-allonseleno-carboxamide,

Ethyl-2-(2,3,5-tri-O-benzoyl-2-β-D-ribofuranosyl)-selenazole-4-carboxylate.

The latter two compounds exemplify key compounds in the context of theinvention that serve for the creation of the uniqueselenazolecarboxamide nucleus of Structure I.

The compound 2-β-D-ribofuranosylselenazole-4-carboxamide, hereinaftersometimes referred to as COMPOUND 1, has been shown to exhibitsignificant antitumor activity in vivo and significant antiviralactivity in vitro. The present invention in one aspect relates tocompositions containing this compound and/or ester derivatives ofStructure I in treating tumors in warm blooded animals. According tothis aspect of the invention, the antitumor properties of2-β-D-ribofuranosylselenazole-4-carboxamide and its related esters areutilized by administering to a warm blooded animal an effective amountof a pharmaceutical composition containing as the active ingredient atleast about 0.1 percent by weight, based on the total weight of thecomposition of at least one compound of Structure I.

The present invention in another aspect relates to compositionscontaining compound 1 and/or ester derivatives of Structure I intreating viral infection in warm blooded animals. According to thisaspect of the invention, the antiviral properties of2-β-D-ribofuranosylselenazole-4-carboxamide and its related esters areutilized by administering to a warm blooded animal an effective amountof a pharmaceutical composition containing as the active ingredient atleast about 1.0 percent by weight, based on the total weight of thecomposition of at least one compound of Structure I.

Specifically noted for R₁, R₂, and R₃ of the compounds of the invention,as preferred acyl groups, are acetyl, propionyl, butyryl, isobutyryl andbenzoyl. Noted as preferred salts when R₃ is phosphono are the alkalimetals and ammonium or substituted ammonium salts such as the sodium,potassium or ammonium salt.

Preferably, when R₁ and R₂ are each H, R₃ is H, C₁ -C₈ acyl or ##STR3##and when R₁ and R₂ are each C₁ -C₈ acyl, R₃ is C₁ -C₈ acyl.

Pharmaceutical compositions of the invention can be formulated in anysuitable way, preferably with an inert carrier. Preferably, thepharmaceutical carrier is chosen to allow administration of a suitableconcentration of the composition of the invention as a solution orsuspension by injection into an afflicted warm blooded animal. Dependingon the host harboring the tumor, the type of tumor, and the tumor site,or, depending on the viral infection and type and site of infection, asthe case may be, administration by injection may be intravenous,intramuscular, intracerebral, subcutaneous, or intraperitoneal.

Alternatively, the composition of the invention may be formulated in anappropriate pharmaceutical carrier allowing for administration byanother route such as oral, ophthalmic, topical or by suppository.

The acyl groups can be selected from a group consisting of straightchain, branch chain, substituted, unsaturated, saturated or aromaticacids such as, but not necessarily limited to, acetic, trifluoroacetic,propionic, n-butyric, isobutyric, valeric, caproic, pelargonic,enanthic, caprylic, lactic, acrylic, propargylic, palmitic, benzoic,phthalic, salicylic, cinnamic and naphthoic acids. With respect tophosphate compounds of the invention, the phosphoryl ester can be as afree acid or as a salt form. Acceptable salts of the phosphate moietycan be selected from, but not necessarily limited to, a group consistingof alkali and alkaline earths, e.g., sodium, potassium, calcium,magnesium and lithium; ammonium and substituted ammonium, includingtrialkylammonium, dialkylammonium and aklylammonium, e.g.,triethylammonium, trimethylammonium, diethylammonium, octylammonium andcetyltrimethylammonium; and cetylpyridinium.

The invention in another aspect relates to a process for the productionof ribofuranosylselenazole-4-carboxamide compounds having the describedStructure 1. The process comprises:

(a) subjecting an alkyl2-(2,3,5-tri-O-acyl-β-D-ribofuranosyl)selenazole-4-carboxylate toammonolysis,

(b) phosphorylating 2-β-D-ribofuranosylselenazole-4-carboxamide or a2'-O-acyl, 3'-O-acyl or 2', 3'-di-O-acyl derivative thereof,

(c) acylating 2-β-D-ribofuranosylselenazole-4-carboxamide,

(d) de-isopropylidenating2-(5-O-acyl-2,3-O-isopropylidene-β-D-ribofuranosyl)selenazole-4-carboxamide,or

(e) de-tritylating 2-(2-O-acyl-, 3-O-acyl-, or 2,3-di-acyl-5-O-triphenylmethyl-β-D-ribofuranosyl)-selenazole-4-carboxamide.

The ammonolysis reaction, for the production of2-β-D-ribofuranosylselenazole-4-carboxamide (COMPOUND 1), is carried outin a suitable solvent such as methanol. The reaction conditions can bevaried as for example at ambient temperature and pressure, andpreferably at room temperature until the reaction is complete, e.g., forabout 24 hours. The product is isolated from the reaction mixture in anysuitable way such as by column chromatography. The alkyl and acyl groupsof the starting material can be varied widely since they are removed inthe reaction and thus their choice is not critical. Preferred alkylgroups are C₁ -C₈ alkyl groups. Preferred acyl groups are acetyl,n-butyryl, and benzoyl.

The phosphorylation reaction, for the production of2-β-D-ribofuranosylselenazole-4-carboxamide 5'-phosphate compounds, iscarried out with the mentioned COMPOUND 1 (or a 2'-O-acyl, 3'-O-acyl, or2',3-di-O-acyl derivative thereof) and a phosphorylating agent such asphosphoryl chloride, advantageously in the cold, in a suitable mediumsuch as triethylphosphate or pyridine and acetonitrile. The product isisolated from the reaction mixture in any suitable way such as byionexchange chromatography. Acylation is carried out by reactingCOMPOUND 1 with the acylating agent such as an acid anhydride orchloride, preferably in excess at ambient temperature until the reactionis complete. The de-isopropylidenation reaction is carried out bytreating the2-(5-O-acyl-2,3-O-isopropylidene-β-D-ribofuranosyl)selenazole-4-carboxamidewith a suitable deprotective agent such as acetic acid, that willselectively remve the isopropylidene group, at elevated temperatureuntil the reaction is complete, e.g. at steam bath temperature for ashort period, and isolating the resulting 5'-O-acyl product by asuitable method such as solvent removal and chromatographic work-up ofthe residue, e.g. using ethyl acetate solvent on silica gel with 20percent (v/v) ethyl acetate in chloroform eluant. The de-tritylationreaction is carried out similarly by treating 2-(2-O-acyl-, 3-O-acyl-,or2,3-di-O-acyl-5-O-triphenylmethyl-β-D-ribofuranosyl)selenazole-4-carboxamidewith a suitable deprotective agent such as acetic acid, that willselectively remove the trityl group, and by isolating the resulting2-O-acyl-, 3-O-acyl-, or2,3-di-O-acyl-β-D-ribofuranosyl)selenazole-4-carboxamide from thereaction mixture by a similar work-up of the residual product aftersolvent removal.

The invention in another aspect relates to a process for the productionof ribofuranosylselenazole-4-carboxamide compounds which comprisesproducing 2,5-anhydro-3,4,6-tri-O-acyl-D-allonselenocarboxamide of thestructure II wherein Z is --C(NH₂)Se by reacting2,3,5-tri-O-acyl-β-D-ribofuranosyl cyanide with liquid hydrogen selenidein the presence of amine catalyst. The catalyst may be adialkylaminopyridine, preferably 4-dimethylaminopyridine. Acyl moieties,as defined above, of the2,5-anhydro-3,4,6-tri-O-acyl-D-allonselenocarboxamide may be any of awide range of alkyl and aromatic acids, preferably acetic or benzoicacids. The reaction is allowed to proceed in a pressure vessel such as asealed bomb, at ambient temperature and pressure for one to 24 hours.The selenocarboxamides are obtained in pure form by venting the excesshydrogen selenide and subjecting the residue to extraction andchromatography.

The invention in still another aspect relates to a process for theproduction of ribofuranosylselenazole-4-carboxamide compounds whichcomprises cyclizing2,5-anhydro-3,4,6-tri-O-acyl-D-allonselenocarboxamide with a lower (C₁-C₈) alkyl or aralkyl bromopyruvate(ROCOCOCH₂ Br) to obtain an alkyl oraralkyl 2-(2,3,5-tri-O-acyl-β-D-ribofuranosyl)selenazole-4-carboxylateof the structure II wherein Z is a selenazol-4-(lower alkyl oraralkyl)carboxylate-2-yl group. The cyclization reaction is carried outin the cold in a suitable solvent such as acetonitrile or a low boilingalcohol.

The invention and the best mode of carrying out the same are describedin the following illustrative examples.

EXAMPLE 1 2-β-D-Ribofuranosylselenazole-4-Carboxamide, COMPOUND 12,5-Anhydro-3,4,6-Tri-O-Benzoyl-D-Allonselenocarboxamide

(a) A mixture of 2,3,5-tri-O-benzoyl-β-D-ribofuranosylcyanide (10.0g,21.2 mmol), 4-dimethylaminopyridine (200 mg) and liquid hydrogenselenide(condensed under N₂ atmosphere, 20 ml) was stirred in a sealed bomb atroom temperature for 20 hours. Hydrogen selenide was allowed toevaporate. The dark colored residue was dissolved in chloroform (200 ml)and washed successively with water (3×50 ml), saturated NaHCO₃ (3×50 ml)followed by water (2×50 ml). The chloroform portion was dried (MgSO₄)and evaporated under vacuum to provide the subtitle product as a foam inalmost quantitative yield. The latter product of analytical purity wasprovided by column chromatography (silica gel, 5 percent ethyl acetatein chloroform). The product developed a purple color when the silica gelchromatogram of the product was sprayed with a dilute ethanolic solutionof 2,3-dichloronaphthoquinone and exposed to ammonia. Analysiscalculated for C₂₇ H₂₃ NO₇ Se: C, 58.91; H, 4.21; N, 2.54; Se, 13.98.Found: C, 58.81; H, 4.29: N, 2.51; Se, 13.74.

Reaction of 2,5-Anhydro-3,4,6-Tri-O-Benzoyl-D-Allonseleno-carboxamidewith Ethyl Bromopyruvate and the Snythesis of Ethyl2-(2,3,5-Tri-O-Benzoyl-D-Ribofuranosyl)selenazole-4-Carboxylates

(b) A solution of2,5-anhhydro-3,4,6-tri-O-benzoyl-D-allonselenocarboxamide (5.5 g, 10mmol) in acetonitrile (60 ml) was cooled in ice. Ethylbromopyruvate (3.0g) in acetonitrile (20 ml) was added dropwise (10 minutes). The ice bathwas removed and the reaction mixture was stirred at room temperature forone hour. The solvent was evaporated in vacuo and the residue wastriturated with a saturated sodium bicarbonate solution (100 ml) andextracted with ethyl ether (2×100 ml). The combined ether portion waswashed with water and dried (MgSO₄). The ether was evaporated in vacuoand the residue (syrup) was passed through a silica gel (300 g) columnpacked in chloroform. Elution with 5 percent ethyl acetate in chloroformprovided subtitle products: namely the fast moveing ethyl2-(2,3,5-tri-O-benzoyl-2-β-D-ribofuranosyl)selenazole-4-carboxylate (2.5g) and the slow moving ethyl2-(2,3,5-tri-O-benzoyl-2-β-D-ribofuranosyl)selenazole-4-carboxylate (1.0g) isolated after evaporation under reduced pressure as thick syrups.The beta isomer, ethyl 2-(2,3,5-tri-O-benzoyl-2-β-D-ribofuranosyl)selenazole-4-carboxylate, is characterized by anoptical rotation, 1.07 percent in methanol, [α]_(D) ²⁵ =-34 7°. Analysiscalculated for C₃₂ H₂₇ NO₉ Se (648.51): C, 59.26; H, 4.20; N, 2.16.Found: C, 59.44; H, 4.21; N, 1.89.

COMPOUND 1

(c) Ethyl2-(2,3,5-tri-O-benzoyl-β-D-ribofuranoysl)selenazole-4-carboxylate (3.2g, 5 mmol) was dissolved in methanol (100 ml), cooled and saturated withammonia (0 degrees C.). The solution was stirred in a pressure bottle atroom temperature for 48 hours. The solvent was evaporated in vacuo andthe residue was extracted with chloroform (25 ml×3). The chloroformportion was discarded. The residue was adsorbed on silica gel (10 g)with the help of methanol and applied on a silica gel column (2.8×45 cm)packed in ethyl acetate. The column was eluted with solvent E (ethylacetate, n-propanol, H₂ O; 4:1:2; v/v; top layer provides solvent E) andthe homogeneous fractions (Rf=0.42, silica gel tlc in solvent (E)containing the major product were collected. The solvent was evaporatedin vacuo and the title product as a residue was crystallized from2-propanol: yield 900 mg of the title product, COMPOUND 1, (60 percent)mp 135-136 degrees C. The residue provided a second crop (200 mg) withmp 131-133 degrees C. Analysis calculated for C₉ H₁₂ N₂ O₅ Se: C, 35.19;H, 3.94; N, 9.12; Se, 25.71. Found: C, 335.43; H, 3.97; N, 9.03; Se,25.55; [α]_(D) ²⁵, 1.07 percent in methanol, -22.2 degrees.

EXAMPLE 2 2-β-D-Ribofuranosylselenazole-4-Carboxamide 5'-phosphate,COMPOUND 2

Water (151 mg, 8.4 mmol) was added carefully to a solution (maintainedat 0 degrees C. by stirring) of phosphoryl chloride (2.0 g, 13.2 mmol),pyridine (1.21 g, 14.4 mmol) and acetonitrile (2.3 g, 56.7 mmol).2-β-D-Ribofuranosylselenazole-4-carboxamide (921 mg. 3.0 mmol) was addedto the solution and the reaction mixture was stirred for 4 hours at 0degrees C. A clear solution was obtained which was poured into ice water(50 ml) and the pH was adjusted to 2.0 with concentrated sodiumhydroxide. The solution was applied to a column of activated charcoal(30 g.), and the column was washed thoroughly with water until theeluate was salt free. The column was eluted with a solution ofethanol-water-concentrated ammonium hydroxide (10:10:1) and thefractions (25 ml each) were collected. The fractions containing thetitle nucleotide product, COMPOUND 2, in pure form (tlc, silica gel,acetonitrile-0.1 N ammonium chloride (7:3) were collected and evaporatedto dryness under vacuum. The anhydrous residual product, COMPOUND 2, wasdissolved in water and passed through a column of Dowex 50W-X8 (20-50mesh, H⁺ form, 15 ml). The column was washed with water and the fractioncontaining the nucleotide was collected. The solution was concentratedto a small volume (5 ml) and passed through a column of Dowex 50W-X8(20-50 mesh, Na⁺ form, 15 ml). The column was washed with water. Thefraction containing the nucleotide as the sodium salt was lyophilized.The residue was triturated with ethanol collected by filtration anddried (P₂ O₅), to provide 580 mg (42 percent) of2-β-D-ribofuranosylselenazole-4-carboxamide 5'-phosphate as monosodiumtrihydrate in the crystalline form. Analysis calculated for C₉ H₁₂ N₂ O₈PSeNa.3H₂ O: C, 23.33; H, 3.90; N, 6.05; P, 6.69; Se, 17.04. Found: C,23.01; H, 3.76; N, 5.86; P, 7.02; Se, 16.32.

EXAMPLE 32-(2,3,5-Tri-O-Acetyl-β-D-Ribofuranosyl)selenazole-4-Carboxamide,COMPOUND 3

A mixture of 2-β-D-ribofuranosylselenazole-4-carboxamide (1.0 g, 3.25mmol), N,N-dimethylaminopyridine (catalyst, 80 mg) and acetic anhydride(15 ml) was stirred at room temperature for 3 hours. The solvent wasevaporated in vacuo and coevaporated with water (10 ml×2) to provideCOMPOUND 3 as a white crystalline product which was triturated withwater and collected by filtration. The product was recrystallized fromwater containing a few drops of ethanol to provide white needles: yield1.2 g (85 percent), mp 117-119 degrees C. Analysis calculated for C₁₅H₁₈ N₂ O₈ Se: C, 41.58; H, 4.19; N, 6.47; Se, 18.22. Found: C, 41.80; H,4.30; N, 6.58; Se, 17.97. Corresponding 2', 3', 5'-O-acyl compounds ofthe invention are prepared from COMPOUND 1 by this procedure by reactingthe latter with the appropriate acid anhydride until the reaction iscomplete and isolating the product in pure form.

EXAMPLE 4 2-(5-O-Acetyl-β-D-ribofuranosyl)selenazole-4-carboxamide

2-β-D-ribofuronosylselenazole-4-carboxamide is first isopropylidenatedwith 2,2-dimethoxypropane, 70 percent perchloric acid and acetone toselectively protect the 2',3'-hydroxyls and then the 5'-hydroxyl isacetylated with acetic anhydride in pyridine as in Example 3 to provide2-(5-O-acetyl-2,3-O-isopropylidene-β-D-ribofuranosyl)selenazole-4-carboxamide.Selective removal of the acid sensitive isopropylidene group with 80percent acetic acid and purification by chromatography provides2-(5-O-acetyl-β-D-ribofuranosyl)selenazole-4-carboxamide as acrystalline solid.

EXAMPLE 5 2-(2-O-Acetyl-β-D-ribofuranosyl)selenazole-4-carboxamide and2-(3-O-Acetyl-β-D-ribofuranosyl(selenazole-4-carboxamide

2-β-D-ribofuranosylselenazole-4-carboxamide in pyridine is successivelytreated first with one molar equivalent of triphenylmethyl chloride andthen with one equivalent of acetic anhydride to provide a mixture of the5'-O-tritylated title products which, after chromatographicpurification, afford the separate products 2-(2- and3-O-acetyl-5-O-triphenylmethyl-β-D-ribofuranosyl)selenazole-4-carboxamideas pure oils. Detritylation of2-(2-O-acetyl-5-O-triphenylmethyl)-selenazole-4-carboxamide with 80percent acetic acid affords2-(2-O-acetyl-β-D-ribofuranosyl)selenazole-4-carboxamide as acrystalline solid.

2-(3-O-Acetyl-β-D-ribofuranosyl)selenazole-4-carboxamide in crystallineform is prepared in like manner from the corresponding 5'-O-tritylatedproduct.

EXAMPLE 6 2-(2,3-Di-O-acetyl-β-D-ribofuranosyl)selenazole-4-carboxamide

2-(5-O-Triphenylmethyl-β-D-ribofuranosyl)selenazole-4-carboxamideprepared by the procedure of Example 5 in which COMPOUND 1 in pyridineis treated with an equivalent of triphenylmethyl chloride is treatedwith excess acetic anhydride in pyridine. The resulting2',3'-di-O-acetyl product is isolated and detritylated as in Example 5.Chromotagraphic purification of the reaction mixture provides the titleproduct, 2,3-di-O-acetyl-β-D-ribofuranosyl)selenazole-4-carboxamide inpure form as a hard foam.

EXAMPLE 7 2-(5-O-Butyrl-β-D-ribofuranosyl)selenazole-4-carboxamide and2-(5-O-Benzoyl-β-D-ribofuranosyl)selenazole-4-carboxamide.

Each of these 5'-acid esters is prepared in a fashion analogous to theprocedure of Example 4 using COMPOUND 1 as a starting material. Thus thestarting material is 2',3'-O-isopropylidenated and the 5'-hydroxyl groupof the resulting 2',3'-O-isopropylidene compound is monoacylated usingn-butyric anhydride and benzoic anhydride, respectively. Selectiveremoval of the acid sensitive isopropylidene group with 80 percentacetic acid and purification by chromatography provides each of therespective title compounds in pure form.

EXAMPLE 8

2-(2-O-Butyryl-β-D-ribofuranosyl)selenazole-4-carboxamide,

2-(2-O-Benzoyl-β-D-ribofuranosyl)selenazole-4-carboxamide,

2-(3-O-Butyryl-β-D-ribofuranosyl)selenazole-4-carboxamide,

2-(3-O-Benzoyl-β-D-ribofuranosyl)selenazole-4-carboxamide

Each of these acid esters of 2-β-D-ribofuranosylselenazole is preparedby the procedure illustrated in Example 5. Thus COMPOUND 1 in pyridineis 5'-O-tritylated, the resulting O-trityl compound is monoacylated witheither n-butyric anhydride or benzoic anhydride as required, and theresulting mixture of 2'-and 3'-O-acyl-5-O-trityl products subjected tochromatographic purification to provide the separate 2-(2- and3-O-acyl-5-O-triphenylmethyl-β-D-ribofuranosyl)selenazole-4-carboxamidesin pure form. Detritylation of the respective 2-(2- or3-O-acyl-5-O-triphenylmethyl)selenazole-4-carboxamide with 80 percentacetic acid gives the corresponding title product as a crystallinesolid.

EXAMPLE 9 2-(2,3-Di-O-butyryl-β-D-ribofuranosyl)selenazole-4-carboxamideand 2-(2,3-Di-O-benzoyl-β-D-ribofuranosyl)selenazole-4-carboxamide

Each of these di-acid esters of2-β-D-ribofuranosylselenazole-4-carboxamides is prepared from2-(5-O-triphenylmethyl-β-D-ribofuranosyl)selenazole-4-carboxamide usingexcess n-butyric anhydride and benzoic anhydride, respectively, inpyridine by the procedure described in Example 6.

EXAMPLE 102-(2-O-Acetyl-5-O-phosphono-β-D-ribofuranosyl)selenazole-4-carboxamide

2-(2-O-Acetyl-β-D-ribofuranosyl)selenazole-4-carboxamide is treated with1 to 5 equivalents of phosphoryl chloride in the presence oftriethylphosphate at 0 decrees C. After complete dissolution isobtained, the solution is poured over crushed ice and adjusted withsodium hydroxide solution to pH 7, extracted with chloroform, and placedon a column of ion exchange resin (Dowex AG 1×8, formate). The column isfirst washed with water, then the product is eluted with a gradient ofwater-formic acid. The fractions containing pure product are pooled andevaporated to dryness. The residue is recrystallized from ethanol-waterto provide the free acid of the title compound as a crystalline solid.

EXAMPLE 11

2-(2-O-Butyryl-5-O-phosphono-β-D-ribofuranosyl)selenazole-4-carboxamide

2-(2-O-Benzoyl-5-O-phosphono-β-D-ribofuranosyl)selenazole-4-carboxamide

2-(3-O-Butyryl-5-O-phosphono-β-D-ribofuranosyl)selenazole-4-carboxamide

2-(3-O-Benzoyl-5-O-phosphono-β-D-ribofuranosyl)selenazole-4-carboxamide

2-(2,3-Di-O-acetyl-5-O-phosphono-β-D-ribofuranosyl)selenazole-4-carboxamide

2-(2,3-Di-O-butyryl-5-O-phosphono-β-D-ribofuranosyl)selenazole-4-carboxamide

2-(2,3-Di-O-benzoyl-5-O-phosphono-β-D-ribofuranosyl)selenazole-4-carboxamide

The title compounds in pure form are each prepared by the proceduredescribed in Example 10 by treating the corresponding 5'-hydroxylcompound with phosphoryl chloride in the presence of triethyl phosphateat 0 degrees C.; followed by appropriate work-up of the reactionmixture, as described, to provide the free acid as a crystalline solid.

As illustrative examples of the antitumor use of COMPOUNDS 1 and 2,Examples 12 through 14, below, are given. In these examples, theefficacy of the compounds is demonstrated using standard tests against atumor, Lewis lung carcinoma. The tests utilized in these illustrativeexamples were conducted by the Developmental Therapeutics Program,Division of Cancer Treatment, National Cancer Institute. The tests weresupervised by this agency utilizing their standard protocols andprocedures. All tests conformed to these protocols and all tests wereevaluated under the criteria defined by these protocols. The followingrepresentative examples illustrate confirmed activity of theillustrative compounds of the invention against screening tumor systemsof the National Cancer Institute.

For purposes of the following examples, the abbreviation IP stands forintraperitoneal and IV stands for intravenous. The mean and mediansurvival times are calculated in instruction 14 (revised 6/78) of theScreening Data Summary, Developmental Therapeutics Program, Division ofCancer Treatment, National Cancer Institute. The contents of thisScreening Data Summary including appropriate revisions are hereinincorporated by reference.

In the illustrated examples below, the vehicle used as carrier for thedrug was injected (minus any drug therein) into the control animals atthe same level of use of the vehicle in the drug treated animals inorder to eliminate any vehicle effect of the tests.

2-β-D-Ribofuranosylselenazole-4-carboxamide, COMPOUND 1, is indicated asbeing active against Lewis lung carcinoma as per Example 12 andsuccessfully passed the DN (decision network) 2 criteria of the NationalCancer Institute Testing. For example 12, B₆ D₂ F₁ male mice were usedand challenged with Lewis lung carcinoma. The median survival time ofthe test animals was compared to that of appropriate control animals.Based on this criterion COMPOUND 1 was considered as an effectiveantitumor agent.

In Example 12, forty control animals and ten test animals were used,each at dose levels indicated below in Table 1. For both the controlgroup and the drug treated group, tumors were induced by IV injection onday zero followed by initiation of drug treatment on day one. ForExample 12 water was used as the drug vehicle.

In both the control group and the drug treated group in Example 12, theanimals were inoculated on day zero with a homogenate of 10⁶ seed cellsof Lewis lung carcinoma. For Example 12, drug treatment was started onday one and COMPOUND 1 given once daily for nine days. Day five wasutilized as the cut-off date for deaths attributable to toxicity of thedrug. There was no mortality attributable to drug toxicity in thisexample. Efficacy of treatment was determined by comparing mediansurvival time of the control animals and is expressed as percentageincrease of treated animals/control animals (T/C).

The test period was for sixty days and at the end of the sixty dayperiod all animals surviving in the test groups were evaluated as eithercured, no-takes, or tumor survivors.

EXAMPLE 12

In this example, the drug tested animals were injected IP with the doselevel noted in Table I below. Ten animals were treated at each doselevel. No control animals survived beyond day 23 with a median deathdate of day 19.0. At dose levels of 75 and 13 mg/kg, all of the treatedanimals survived, giving a T/C of 315 percent and 7 out of 10 and 5 outof 10 cures, respectively. Furthermore, at the relative low dose of 3mg/kg, DN2 criteria of NCI were passed by achievement of T/C of 152percent. One cure was achieved at this dose level.

COMPOUND 1 is indicated as being an active antitumor agent in themultiple dose studies noted.

                  TABLE 1                                                         ______________________________________                                        Antitumor Activity - COMPOUND 1                                                                 Control                                                     Drug              Group           Percent                                     Dose  Treated Group                                                                             Survival        Treated Animals/                            mg/kg Survival Time                                                                             Time      Cures Control Animals                             ______________________________________                                        75    60.0        19.0      7/10  315%                                        50    18.0                  1/10   94%                                        33    18.0                  3/10   94%                                        20    16.0                  2/10                                              13    60.0                  5/10  315%                                         9    10.4                  3/10                                               6    12.0                  1/10                                               3    28.9                  1/10  152%                                        ______________________________________                                    

As is shown in Table I, COMPOUND 1 shows outstanding activity againstLewis lung carcinoma. Lewis lung carcinoma is an excellent example of ametastatic tumor system. The test animals and control animals of Example12 were inoculated IV with a homogenate of the tumor. Dramaticexpression of this tumor is then seen in the lungs. The ability tometastasize is a property that uniquely characterizes a malignant tumorfrom a benign tumor.

EXAMPLE 13

In this example COMPOUND 1 was tested in B₆ D₂ F₁ female mice challengedwith Lewis lung carcinoma. The median survival time of the test animalswas compared to that of appropriate control animals. Based on thiscriterion COMPOUND 1 was found to be an effective antitumor agent asshown in Table II. At dose levels of 50, 25, and 12.50 mg/kg all of thetest animals survived, giving a T/C of 355 percent; 6 out of 10, 7 outof 10, and 8 out of 10 cures were achieved, respectively.

                  TABLE II                                                        ______________________________________                                        Antitumor Activity - COMPOUND 1                                                                 Control                                                     Drug              Group           Percent                                     Dose  Treated Group                                                                             Survival        Treated Animals/                            mg/kg Survival Time                                                                             Time      Cures Control Animals                             ______________________________________                                        200   10.7        16.9      0/10                                              100   20.3                  3/10  120                                         50    60.0                  6/10  355                                         25    60.0                  7/10  355                                         12    60.0                  8/10  355                                         ______________________________________                                    

COMPOUND 1 was further tested in a Lewis lung in vivo mouse model usingB₆ D₂ F₁ female mice challenged with Lewis lung carcinoma. The reultsare set forth in Table III. As shown, COMPOUND 1 at low dose levels (24,12 and 6 mg/kg) had a T/C of 297 percent; 9 out of 10, 9, out of 10, and5 out of 10 cures were achieved, respectively. COMPOUND 1 achieved DN2level activity (percent T/C greater than 150) of 153 at the low dose of3 mg/kg. The results confirm that COMPOUND 1 is an effective antitumoragent for Lewis lung carcinoma in mice.

                  TABLE III                                                       ______________________________________                                        Antitumor Activity - COMPOUND 1                                                                 Control                                                     Drug              Group           Percent                                     Dose  Treated Group                                                                             Survival        Treated Animals/                            mg/kg Survival Time                                                                             Time      Cures Control Animals                             ______________________________________                                        24.00 60.0        20.2      9/10  297                                         12.00 60.0                  9/10  297                                         6.00  60.0                  5/10  297                                         3.00  31.0                  0/10  153                                         1.50  22.3                  0/10  110                                         0.75  21.7                  0/10  107                                         ______________________________________                                    

EXAMPLE 14

COMPOUND 2, 2-(5-phosphono-β-D-ribofuranosyl)selenazole-4-carboxamidewas screened for Lewis lung carcinoma activity in a manner similar tothat described in Example 12. In this example, B6_(D) 2_(F) 1 male micewere challenged with Lewis lung carcinoma. The mean survival time of thetest animals was compared to that of appropriate control animals. Basedon this criterion, COMPOUND 2 was considered to be an effectiveantitumor agent. In this example, cures ranging from 1 out of 10 to 4out of 10 treated mice were obtained at a dose ranging from 9 mg/kg to75 mg/kg, Table IV below. At 13 mg/kg the treated, surviving mice have aT/C of 315 percent and 4 out of 20 cures were achieved.

                  TABLE IV                                                        ______________________________________                                        Antitumor Activity - COMPOUND 2                                                                 Contorl                                                     Drug              Group           Percent                                     Dose  Treated Group                                                                             Survival        Treated Animals/                            mg/kg Survival Time                                                                             Time      Cures Control Animals                             ______________________________________                                        75    15.5        19.0      1/10                                              50    12.5                  3/10                                              33    12.5                  3/10                                              20    12.0                  2/10                                              13    60.0                  4/10  315                                          9    12.5                  1/10                                               6    22.0                  0/10  115                                          3    20.8                  0/10  109                                         ______________________________________                                    

In further tests, growth-inhibitory effects of COMPOUND 1 and COMPOUND 2were measured against 3 mouse tumor lines in suspension cell culture.Cell cultures were initiated at a density of 50,000 cells/ml in mediumRPMI 1630 supplemented with 10 percent fetal calf serum. Composition ofthis culture medium and details of the culture procedure followed thepublished method (G. E. Moore, A. A. Sandberg and K. Ulrich, SuspensionCell Culture and in vivo and in-vitro chromosome constitution of mouseleukemia L1210, Journal of the National Cancer Institute 36, 405-415,1966). Cultures were maintained at 37 degrees in stationary suspensionculture under a 95 percent air +5 percent CO₂ atmosphere. Test compoundswere added to treated cultures at the time of initiation, and werepresent continually. After 72 hours, 40-fold dilutions of drug-treatedand untreated control cultures were prepared in 0.9 percent NAClsolution, and cells were counted on an electronic particle counter. Theresults of the tests are shown in Table V below. The growth-inhibitoryeffects are expressed as ID₅₀ values, namely, test compound inhibitorydosages or concentrations required to decrease cell count in treatedcultures to 50 percent of the cell count of untreated control cultures.

                  TABLE V                                                         ______________________________________                                        ID.sub.50 Values (Molar) Against Various Tumor Cell Lines                              L-1210    P388      B.sub.16 Melanoma                                ______________________________________                                        COMPOUND 1 4.0 × 10.sup.-7                                                                     2.7 × 10.sup.-7                                                                   3.5 × 10.sup.-6                        COMPOUND 2 5.8 × 10.sup.-8                                                                     6.6 × 10.sup.-8                                                                   7.5 × 10.sup.-6                        ______________________________________                                    

These results show that COMPOUND 1 and COMPOUND 2 both inhibit growth incell culture for representative tumor lines at extremely low dosage. Inthis regard, the 5'-phosphate (COMPOUND 2) appears to be superior toCOMPOUND 1.

EXAMPLE 15

In one embodiment, COMPOUND 1 showed activity against both small andlarge viruses of both DNA and RNA types by the virus rating (VR) methodof Sidewell et al., Appl. Microbiol. 22, 797 (1971). A virus rating thatis greater than 1.0 indicates definite antiviral activity. A virusrating of 0.5-0.9 indicates moderate antiviral activity, and a virusrating smaller than 0.5 suggests slight or no apparent antiviralactivity. The results reported below were obtained by testing onMicrotest II (Falcon Plastics) plastic panels with a monolayer of Veroor HeLa cells.

    ______________________________________                                        ANTIVIRAL ACTIVITY OF COMPOUND 1                                                        Virus Rating                                                                              ED.sub.50 (ug/ml)                                       Virus       Vero   HeLa       Vero   HeLa                                     ______________________________________                                        RNA viruses                                                                   Para 3      2.6    2.2        1      1                                        Measles     2.0    1.8        1      2                                        Mumps       1.7    1.0        5      1                                        Reo 3       1.8    2.7        1      8                                        VSV         0.4    2.1        1000   9                                        Cox B1      1.7    1.8        15     10                                       Cox B4      0.0    2.3        1000   6                                        DNA viruses                                                                   VV          2.1    2.4        3      2                                        Ad-2        --     1.9        --     9                                        HSV-1       1.2    1.4        30     2                                        HSV-2       1.5    1.5        10     4                                        ______________________________________                                    

The results indicte that COMPOUND 1 has good broad spectrum antiviralactivity against both DNA and RNA viruses. From the DNA viruses, therepresentatives of the families Poxviridae (Vaccinia) and Herpesviridae(HSV-1, HSV-2) were inhibited most. Greatest activity was observed inthe representatives of RNA families Paramyxoviridae (Para-3, Mumps,Measles) and Reoviridae (Reo-3). Excellent antiviral activity wasmeasured in Heha cells for the families Adenoviridae (Adeno-2),Picornaviridae (Cox B1, Cox B4) and Rhabdoviridae (VSV).

The studies further indicate that antiviral activity of COMPOUND 1 isboth virucidal (against Vaccinia) and virusstatic (against Para-3 andHSV-1), depending upon the virus and cell line used. Also, theprophylactic use of COMPOUND 1 may enhance its antiviral activityagainst virustatic species such as HSV-1. COMPOUND 1 is non-toxic toVero, Hela and MRC-5 cells in 1000 ug/ml quantities (highestconcentration tested).

These results further show that COMPOUND 1 inhibits viral cytopathiceffects in cell culture for representative viruses at extremely lowdosage; COMPOUND 1 exhibits low cytotoxicity and is soluble in aqueousmedia.

The following representative examples 16 through 20, are given asillustrative pharmaceutical compositions utilizing different carriers.In these examples, example 16 illustrates the use of the compounds ofthe invention in injectables suitable for intravenous or other types ofinjection into the host animal. Example 17 is directed to an oral syruppreparation, Example 18 to an oral capsule preparation and Example 19 tooral tablets. Example 20 is directed to use of the compounds of theinvention in suitable suppositories. For example 16 through 20, theingredients are listed followed by the methods of preparing thecompositions.

EXAMPLE 16

    ______________________________________                                        INJECTABLES                                                                   Example 16a COMPOUND 1                                                        ______________________________________                                        COMPOUND 1     125 mg-500 mg                                                  Water for Injection USP q.s.                                                  ______________________________________                                    

COMPOUND 1 is dissolved in the water and passed through a 0.22 micronfilter. The filtered solution is added to ampoules or vials, sealed andsterilized.

    ______________________________________                                        Example 16b COMPOUND 2                                                        ______________________________________                                        COMPOUND 2 as a Sodium Salt                                                                       125 mg-500 mg                                             Water for Injection USP q.s.                                                  ______________________________________                                    

Prepared as per Example 16a above.

EXAMPLE 17

    ______________________________________                                        SYRUP                                                                         Example 17a COMPOUND 1                                                        ______________________________________                                        250 mg Active ingredient/5 ml syrup                                           COMPOUND 1               25     g                                             Purified Water USP       200    ml                                            Cherry Syrup q.s. or     1000   ml                                            ______________________________________                                    

COMPOUND 1 is dissolved in the water and to this solution the syrup isadded with mild stirring.

    ______________________________________                                        Example 17b COMPOUND 2                                                        ______________________________________                                        125 mg Active Ingredients/5 ml syrup                                          COMPOUND 2 as a Sodium Salt                                                                            25     g                                             Purified Water USP q.s. or                                                                             200    ml                                            Cherry Syrup q.s. ad     1000   ml                                            ______________________________________                                    

Prepared as per Example 17a above.

EXAMPLE 18

    ______________________________________                                        CAPSULES                                                                      Example 18a COMPOUND 1                                                        50 mg, 125 mg or 250 mg                                                       ______________________________________                                        COMPOUND 1               500    g                                             Lactose USP, Anhydrous q.s. or                                                                         200    g                                             Sterotex Powder HM       5      g                                             ______________________________________                                    

Combine COMPOUND 1 and the Lactose in a twinshell blender equipped withan intensifier bar. Tumble blend for two minutes, blend for one minutewith the intensifier bar and then tumble blend again for one minute. Aportion of the blend is then mixed with the Sterotex Powder, passedthrough a #30 screen and added back to the remainder of the blend. Themixed ingredients are then blended for one minute, blended with theintensifier bar for thirty seconds and tumble blended for an additionalminute. Appropriate sized capsules are filled with 141 mg. 352.5 mg or705 mg of the blend, respectively, for the 50 mg., 125 mg and 250 mgcontaining capsules.

    ______________________________________                                        Example 18b COMPOUND 2                                                        50 mg, 125 mg or 250 mg                                                       ______________________________________                                        COMPOUND 2               500    g                                             Lactose USP, Anhydrous q.s. or                                                                         200    g                                             Sterotex Powder HM       5      g                                             ______________________________________                                    

Mix and fill as per Example 18a.

EXAMPLE 19

    ______________________________________                                        TABLETS                                                                       50 mg, 100 mg or 250 mg                                                       ______________________________________                                        COMPOUND 1              250    g                                              Corn Starch NF          200.0  g                                              Cellullose, Microcrystalline                                                                          46.0   g                                              Sterotex Powder HM      4.0    g                                              Purified Water q.s. or  300.0  ml                                             ______________________________________                                    

Combine the corn starch, the cellulose and COMPOUND 1 together in aplanetary mixer and mix for two minutes. Add the water to thiscombination and mix for one minute. The resulting mix is spread on traysand dried in a hot air oven at 50 degrees C. until a moisture level of 1to 2 percent is obtained. The dried mix is then milled with a Fitzmillthrough a #RH2B screen at medium speed. The Sterotex Powder is added toa portion of the mix and passed through a #30 screen, and added back tothe milled mixture and the total blended for five minutes by drumrolling. Compressed tablets of 150 mg. 375 mg and 750 mg respectively,of the total mix are formed with appropriate sized punches for the 50mg, 125 mg or 500 mg containing tablets.

EXAMPLE 20

    ______________________________________                                        SUPPOSITORIES                                                                 Example 20a COMPOUND 1                                                        125 mg. 250 mg or 500 mg per 3 g                                              ______________________________________                                        COMPOUND 1      125    mg     250  mg   500  mg                               Polyethylene Glycol                                                                           1925   mg     1750 mg   1400 mg                               1540                                                                          Polyethylene Glycol                                                                           825    mg     750  mg   600  mg                               8000                                                                          ______________________________________                                    

Melt the Polyethylene Glycol 1540 and the Polyethylene Glycol 8000together at 60 degrees C. and dissolve COMPOUND 1 into the melt. Moldthis total at 25 degrees C. into appropriate suppositories.

    ______________________________________                                        Example 20b COMPOUND 2                                                        125, 250, 500 MG PER 3 G                                                      ______________________________________                                        COMPOUND 2      125    mg     200  mg   500  mg                               Polyethylene Glycol                                                                           1925   mg     1750 mg   1400 mg                               1540                                                                          Polyethylene Glycol                                                                           825    mg     750  mg   600  mg                               8000                                                                          ______________________________________                                    

Prepare as per Example 20a above.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A compound of thestructure II: ##STR4## wherein R₁, R₂ and R₃ are acyl selected from thegroup consisting of benzoyl and C₁ -C₈ acyl; and Z is selenazol-4-alkylcarboxylate-2-yl, alkyl being selected from the group consisting oflower alkyl and lower aralkyl.
 2. A compound according to claim 1 whichis an alkyl or aralkyl2-(2,3,5-tri-O-acyl-2-β-D-ribofuranosyl)selenazole-4-carboxylate.
 3. Acompound according to claim 2 which is ethyl2-(2,3,5-tri-O-benzoyl-2-β-D-ribofuranosyl)selenazole-4-carboxylate. 4.A process for the production of compounds according to claim 1 whichcomprises cyclizing2,5-anhydro-3,4,6-tri-O-acyl-D-allonselenocarboxamide with an alkyl oraralkyl bromopyruvate to obtain an alkyl or aralkyl2-(2,3,5-tri-O-acyl-B-D-ribofuranosyl)selenazole-4-carboxylate of thestructure II wherein Z is a selenazole-4-(alkyl oraralkyl)carboxylate-2-yl group.